GSM6589592: Biological replicate 1 of vibrio with no glycans; Vibrio cholerae; RNA-Seq
We pelleted 50 μL of cells grown with or without mucin glycans at 12,000g in a tabletop centrifuge for 2 min. Cells were generally grown in AKI media under anaerobic conditions unless otherwise indicated. We resuspended cell pellets in 300 μL of 2x Tissue and Cell Lysis Buffer (Lucigen) with 2 μL of proteinase K. Samples were incubated at 65 °C to aid in cell lysis. Next, 175 μL of MPC Protein Precipitation Reagent (Lucigen) was added to the samples, which were spun for 10 min at the maximum speed in a tabletop centrifuge to precipitate out proteins. The supernatant was then mixed with 500 μL of isopropanol and spun down for 15 min at the maximum speed. The resulting pellet of RNA was washed twice with 70% ethanol and centrifuged for 15 min at the maximum speed following each wash. The RNA was then air-dried for 5 min and dissolved in 50 μL of nuclease-free water. To remove contaminating DNA from the RNA preparation, we incubated each sample with 5 μL of Ambion RNA Turbo Buffer and 2 μL of Turbo DNAse (Thermo Fisher) for 30 min at 37 °C. Next, we added 5 μL of Ambion reaction inactivator to each sample to remove DNAse. Samples were spun at the maximum speed for 2 min, and then supernatant containing purified RNA was collected and stored at -20 °C until use. We depleted the purified RNA of ribosomal RNA using the Illumina RiboZero depletion kit. Depleted RNA was submitted to the MIT BioMicro Center for strand-specific library preparation.